We propose to continue our development of metallic nanostructures (MNS) for DNA analysis. During the upcoming cycle we will focus on MNS which are simple to prepare and have near-term applications in DNA analysis. The structures will be designed to be useful for increased sensitivity in surface-based assays and for future uses for high throughput and/or single strand DNA sequencing. Specific Aim 1. We will develop the use of bimetallic nanoholes in metal films for hybridization assays, single molecule detection and sequencing. The nanoholes will provide enhanced single molecule intensities and optical isolation from the bulk sample. Some of the structures will provide spectral separations and intensification of labeled molecules with the possibility of detection with flow conditions. Specific Aim 2. Develop the use of nanoporous metals as substrates for DNA hybridization assays and high throughput single molecule detection under flow conditions. Specific Aim 3. Develop the use of nanoporous alumina decorated with metals for high throughput DNA assays under flow conditions. These structures will be tested for high sensitivity DNA detection and for real-time hybridization assays without washing steps. Additionally, these structures will be tested for single molecule detection under conditions similar to those used for single strand sequencing based on polymerase or exonuclease. All the proposed structures can be available at low costs for high- throughput applications. PUBLIC HEALTH RELEVANCE: DNA analysis is a fundamental component of biological research, sequencing and medical testing. The goal of this project is to increase the sensitivity and decrease the complexity of methods used for all types of DNA measurements. This goal will be accomplished by the development and design of metallic nanostructures which enhance fluorescence intensities and decrease background. Additionally, these metallic structures will bypass the use of more expensive optical components and in some cases improve on what is currently possible using classical optics. The structures we develop will have multiple applications in testing, single nucleotide or oligo detection and high throughput sequencing. We will develop and test three types of structures, nanoholes, nanoporous metals and nanoporous alumina containing metals. Each type of structure provides unique opportunities for new assay formats and can be readily available to other laboratories at low cost.